Agrobacterium-mediated genetic transformation of Coffea arabica (L.) is greatly enhanced by using established embryogenic callus cultures

Abstract

BackgroundFollowing genome sequencing of crop plants, one of the main challenges today is determining the function of all the predicted genes. When gene validation approaches are used for woody species, the main obstacle is the low recovery rate of transgenic plants from elite or commercial cultivars. Embryogenic calli have frequently been the target tissue for transformation, but the difficulty in producing or maintaining embryogenic tissues is one of the main problems encountered in genetic transformation of many woody plants, including Coffea arabica.ResultsWe identified the conditions required for successful long-term proliferation of embryogenic cultures in C. arabica and designed a highly efficient and reliable Agrobacterium tumefaciens-mediated transformation method based on these conditions. The transformation protocol with LBA1119 harboring pBin 35S GFP was established by evaluating the effect of different parameters on transformation efficiency by GFP detection. Using embryogenic callus cultures, co-cultivation with LBA1119 OD600 = 0.6 for five days at 20 °C enabled reproducible transformation. The maintenance conditions for the embryogenic callus cultures, particularly a high auxin to cytokinin ratio, the age of the culture (optimum for 7-10 months of proliferation) and the use of a yellow callus phenotype, were the most important factors for achieving highly efficient transformation (> 90%). At the histological level, successful transformation was related to the number of proembryogenic masses present. All the selected plants were proved to be transformed by PCR and Southern blot hybridization.ConclusionMost progress in increasing transformation efficiency in coffee has been achieved by optimizing the production conditions of embryogenic cultures used as target tissues for transformation. This is the first time that a strong positive effect of the age of the culture on transformation efficiency was demonstrated. Our results make Agrobacterium-mediated transformation of embryogenic cultures a viable and useful tool both for coffee breeding and for the functional analysis of agronomically important genes.

Stable integration of mgfp5 transgenes following Agrobacterium-mediated transformation in Boesenbergia rotunda cell suspension culture

“…Application of the appropriate concentration of hygromycin B not only enabled effective selection of transformed cells but also allowed synchronization between selection and cell growth in different developmental growth phases, i.e. propagation and regeneration of the suspension cells, which usually involve different media combinations (Ribas et al 2011). …”

“…Agrobacterium-mediated transformation has been the most accessible tool with which to introduce heterologous DNA and manipulate gene expression in plants (Ribas et al 2011). Therefore, a reliable transformation and regeneration system with a low degree of chimerism and high regeneration frequency is a prerequisite (May et al 1995;Sági et al 1995).…”

Agrobacterium-mediated genetic transformation of peanut and the efficient recovery of transgenic plants

“…The experiment was replicated twice and each set within a replicate consisted of 45Á59 explants. Data were analyzed statistically and the confidence index was calculated as described by Ribas et al (2011). The early-mturing varieties with relatively small seeds, Huayu 20 and 26, presented the highest shoot regeneration percentage, with respective levels of 62.61 and 61.61% from cotyledon-derived explants, and 71.58 and 76.92% from mesocotyl-derived explants.…”

“…For sonication for 3 and 5 min, the respective percentages of plant regeneration were 56.93 and 41.00%, and the respective transformation efficiencies were 5.45 and 3.50%. Statistical analysis revealed that submerging The confidence index was calculated as described by Ribas et al (2011). The confidence index was calculated as described by Ribas et al (2011).…”

Morphological characteristics and cell viability of coffee plants calli

“…According RIBAS et al (2011), currently, embryogenic callus derived from leaf tissues is the most widely used target tissue for the genetic transformation of C. arabica and different callus stages can affect plant transformation. However, the induction of embryogenic tissues in C. arabica takes longer and is more diffi cult than in the others cultivated species.…”